Abstract
6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine (inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase Cdelta (PKCdelta) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKCdelta (79 kDa) to yield a catalytic fragment (41 kDa). Inhibition of PKCdelta (with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.
Highlights
Marks of this disease are well described, the etiology is largely undefined
The data presented in this study demonstrate that the commonly used Parkinsonian neurotoxin, 6-OHDA, induced neurotoxicity in PC12 cells via extracellular auto-oxidation and consequent oxidative stress
It is predicted that the oxidative products of 6-OHDA initiated intracellular oxidative stress, and the attenuation of this toxicity by catalase implicates hydrogen peroxide as a key mediator of 6-OHDA-induced cell death
Summary
Marks of this disease are well described, the etiology is largely undefined. a number of biochemical processes and molecular mechanisms have been identified as mediators of neuronal cell death in PD. The site of caspase 3 cleavage lies between the regulatory and catalytic domains, and proteolysis induces permanent dissociation of the two domains and constitutive activation of the catalytic domain [22, 23] This mechanism of activation has been shown to occur in response to a range of apoptotic stimuli, both in neuronal and non-neuronal cell models (22, 24 –32). PKC␦ has been shown to be activated in response to a number of dopaminergic neurotoxins, including 1-methyl-4-phenylpyridinium [30, 33] and methylcyclopentadienyl manganese tricarbonyl [32] in the dopaminergic rat N27 cell line and dieldrin in PC12 cells [31] This may suggest that PKC␦ plays a role in neuronal apoptosis in PD. The downstream effectors of mitochondrial dysfunction and caspase activation were investigated, and the possible role of caspase 3-dependent PKC␦ activation in 6-OHDA-induced apoptosis was explored
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